Comparison of PCR assays with direct microscopy for diagnosis of cutaneous leishmaniasis patients reported from Sindh, Pakistan

The objectives of this study was the molecular identification of Leishmania species and evaluate the diagnostic yield of PCR in comparison to the direct microscopy. This study was conducted at Centre for Molecular Genetics, University of Karachi, from March 2007 - December 2011. Samples were taken by means of Saline Aspirate (SA) from 50 patients suspected of cutaneous leishmaniasis (CL) from Dermatology Department, Jinnah Post Graduate Medical College, Karachi, Pakistan from March 2007 - December 2008. Only four samples (8%) had microscopically positive results. Recovery of parasites in cultures was of 09 strains (18%). These strains were maintained in vitro for the purpose of diagnosis by PCR and vaccine preparation. Three strains were vanished due to bacterial contamination. The remaining 06 culture positive samples along with saline aspirate obtained from all 50 patients. Sore were confirmed for the presence of Leishmania species by PCR method. Electrophoresis pattern from each isolates were compared with reference strains for L. major (MRHO/IR/75/ER), L. tropica (MHOM/SU/74/K27) and L. infantum (MHOM/TN/80/IPTI). Hundred percent isolates subjected to PCR, were similar to L. major (MRHO/IR/75/ER) reference strain. All of these PCR products showed a single band of approximately 650bp representing Leishmania major specific Kinetoplast DNA (kDNA) sequences. This 650bp band was of same size as that of the standard identified Leishmania strain. In conclusion CL caused by L. major infection is prevalent in the Sindh province of Pakistan. This study also proves that PCR method is sensitive diagnostic test in comparison to the direct microscopy.